Eccles:NZM Cell Culture: Difference between revisions
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*The NZM melanoma cell lines are cultured in ITS media, which is prepared as follows: | *The NZM melanoma cell lines are cultured in ITS media, which is prepared as follows: | ||
**1 bottle AlphaMEM media (Invitrogen # 12561-072). | **1 bottle AlphaMEM media (Invitrogen # 12561-072). | ||
**1% Insulin-Transferrin-Selenium (ITS) (Roche # 1074547). | **0.1% Roche Insulin-Transferrin-Selenium (ITS) (Roche # 1074547; 100uL/100mL media). | ||
**10% | **5% FCS (Auckland use 5%, but 10% has been used in Dunedin in addition to 5%). | ||
**Sterile filter the ITS and FCS, culture a test flask of media for 3 days to ensure it is free from contamination. | **Sterile filter the ITS and FCS (optional, and of doubtful usefulness without cells in the flask as well, is to culture a test flask of media for 3 days to ensure it is free from contamination). | ||
*Culture the cells at 37°C in 5% CO<sub>2</sub>. | *Culture the cells at 37°C in 5% CO<sub>2</sub>. | ||
Latest revision as of 15:53, 8 March 2010
Back to Eccles Lab > DGG Protocols
Culturing NZM Cell Lines:
- The NZM melanoma cell lines are cultured in ITS media, which is prepared as follows:
- 1 bottle AlphaMEM media (Invitrogen # 12561-072).
- 0.1% Roche Insulin-Transferrin-Selenium (ITS) (Roche # 1074547; 100uL/100mL media).
- 5% FCS (Auckland use 5%, but 10% has been used in Dunedin in addition to 5%).
- Sterile filter the ITS and FCS (optional, and of doubtful usefulness without cells in the flask as well, is to culture a test flask of media for 3 days to ensure it is free from contamination).
- Culture the cells at 37°C in 5% CO2.
- Most of the NZM stocks we have in liquid nitrogen have some level of contamination. They came like this from Auckland, so don't despair if you see bugs in your flasks, it wasn't you!
- The level of contamination usually stays at a manageable level and the cells grow happily enough. Occasionally it can take off (especially for NZM46 I've noticed) and the cells cannot be saved, but this doesn't happen often.
- The morphology of the cell lines varies, with some being more epithelial-like and others more fibroblastic-like. Some are almost transparent and are very hard to see through a microscope.
- The growth rates also vary, with most lines requiring passaging 2 times a week.
- A 1:4 split is usually sufficient.
